Organisms are constantly adapting and evolving in response to internal and external stimuli. These changes can arise by either new mutations or by genetic variation already present in the system. The latter source of standing genetic variation, which does not affect the usual range of phenotypes, but has a potential to modify a phenotype in the event of an environmental or genetic perturbation, is referred to as cryptic genetic variation (CGV). Due to the environmental and cultural changes in recent human history, the uncovering of CGV could be a potential explanation for the rising incidences of diseases of modernity (i.e. asthma, diabetes, depression, etc.). We believe that cryptic variants are quite prevalent in the genome and do play a significant part in determining the consequences of genetic perturbations in an organism. To demonstrate the release of CGV, we will use the fruit fly, Drosophila melanogaster. In particular, we will investigate how cryptic genetic variation can affect female differentiation in Drosophila melanogaster by perturbing intersex (ix), which encodes a sex-specifically acting component of the Mediator transcriptional regulatory complex. We hypothesize that mutating ix in different genetic backgrounds of flies will reveal varying phenotypic responses, which is a proxy to the release of CGV. By quantifying the amount of variation in perturbed phenotypic responses, we can assess the extent and nature of CGV. To investigate our hypothesis, we transgenically create a new loss-of-function mutation tagged with GFP for ix and introgress this mutation via backcrossing into the genetic backgrounds of the 192 Drosophila Genetic Reference Panel lines. We will measure five quantitative female traits associated with ix (i.e. number of gonopod thorn bristles, proportion width of the sixth tergite that is darkly pigmented, number of bristles on the last transverse row on the foreleg basitarsus, number of spermathecae, and fusion of the anal plates) and analyze the phenotypic variation within and between the lines as well as with and without the introgressed mutation. We will also test for the role of ix in promoting robustness against developmental noise (i.e. microenvironmental variation) since Mediator components have been known to confer robustness against this type of variation in other organisms. In addition to morphological phenotypes, we will also investigate gene expression profiles using microarrays from the pupal genital discs of a subset of the DGRP lines with and without the ix mutation to understand line-by-genotype interactions in gene expression as well as explore correlations between these interactions with the phenotypic variation in the five female traits we will have measured in the first part of the project. With the completion of the project, we will not only hav a better understanding of the prevalence and behavior of cryptic genetic variation, but will have also produced a systematic protocol to study CGV that is highly customizable. We will have created a new strategy for introducing dominantly marked loss- of-function mutations into any gene of choice and a set of balancer DGRP lines as well as a pipeline for data analysis for the general research community to use for future studies.